Multi-formed collagenous biomaterial medical device

ABSTRACT

The invention involves a submucosa tissue that has the capability of being shape formed or shape configured. The submucosa involves a purified form of submucosa tissue. Optionally, the submucosa can be packaged in such a manner to permit sterility or maintain sterility of the submucosa.

STATEMENT OF RELATED APPLICATIONS

[0001] This application claims priority to U.S. application Ser. No.08/916,490, filed Aug. 22, 1997, which claims priority to U.S.provisional application serial No. 60/024,542 (filed Aug. 23, 1996) and60/024,693 (filed Jun. 09, 1996); and to U.S. provisional serial No.60/110,407 filed Dec. 01, 1998.

TECHNICAL FIELD

[0002] The invention generally relates to a medical device and inparticular, to a medical device comprising a collagenous biomaterial.

BACKGROUND OF THE INVENTION

[0003] A naturally occurring biomaterial for medical implantation ismore desirable that a synthetic implant. Synthetic implants tend tocause adverse reactions in a patient, including thrombosis, immuneresponses, and potentially restenosis in vascular applications.Therefore, a medical implant that reduces or eliminates these problemsis a technical advance.

[0004] Collagenous biomaterials are known to be used in medicalapplications as medical devices. As a naturally occurring biomaterial,the implant produces less complications than a synthetic implant.Collagen is used as an abundant source of protein, and is most notablyderived from bovine skin. Collagen forms a matrix that is useable as animplant. However, as a biomaterial, it does not have good manipulationproperties, unless treated in other ways. In addition, one problem withthese material is that collagen biomaterials also carry with themantigens which cause an immune response in the patient. Therefore, aproduct that behaves like collagen in vivo yet is highly manipulativeand elicits less to no negative immune response is a technicalachievement.

SUMMARY OF THE INVENTION

[0005] The foregoing problems are solved and a technical advance isachieved with the present invention. A new biomaterial comprising thesubmucosa of a tissue was discovered to have greater benefit than usingcollagen. For example, the submucosa is shown to exhibit moreremodeling, regrowth, and regeneration of tissue upon implant. It hasbeen shown that submucosal tissue is absorbed by the patient and thusthe patient does not require post-implantation procedures to remove theimplant. The submucosal tissue has been shown to elicit favorable immuneresponse that leads to an accommodation of the submucosal implant versusa rejection based response. Therefore, to further improve the submucosaltissue's industrial utility, the applicants have discovered that thisutility can be achieved by improving the submucosa's purity and formingthe submucosal tissue into various forms. Such forms, include, but arenot limited to sheets, sponges, fluidized, etc. The present inventionrelates to a purified form of submucosal tissue that is treated in sucha manner as to confer some shape memory and shape configuration to theimplant. However, the submucosal tissues are not limited to implants andcan be formed to be used in topical applications as well, such as wounddressings or wound plugs.

[0006] In addition, the problem of maintaining the sterility of themedical device is solved by including the medical device in a pouch orplurality of pouches. The pouches can be at least one of a gaspermeable, sealed, hermetically sealed, sterile UV protected, andmultiple pouched. In addition, the foregoing problems are solved byincluding processes of making the medical device.

BRIEF DESCRIPTION OF THE DRAWING

[0007]FIG. 1 shows two aspects of the invention as the biomaterialmultilaminate sheet or in sponge forms.

[0008]FIG. 2A shows one aspect of the invention as the biomaterial in adouble pouched package, wherein the package is in an open configuration.

[0009]FIG. 2B shows one aspect of the invention as the biomaterial in aclosed double pouched package.

DETAILED DESCRIPTION

[0010] In the discussions herein, a number of terms are used. In orderto provide a clear and consistent understanding of the specification andclaims, the following definitions are provided.

[0011] Bioburden—refers to the number of living microorganisms, reportedin colony-forming units (CFU), found on and/or in a given amount ofmaterial. Illustrative microorganisms include bacteria, fungi, and theirspores.

[0012] Disinfection—refers to a reduction in the bioburden of amaterial.

[0013] Sterile—refers to a condition wherein a material has a bioburdensuch that the probability of having one living microorganism (CFU) onand/or in a given section of the material is one in one-million or less.

[0014] Pyrogen—refers to a substance which produces febrile responseafter introduction into a host.

[0015] Endotoxin—refers to a particular pyrogen which is part of thecell wall of gram-negative bacteria. Endotoxins are continually shedfrom the bacteria and contaminate materials.

[0016] Purification—refers to the treatment of a material to remove oneor more contaminants which occur with the material, for instancecontaminants with which the material occurs in nature, and/ormicroorganisms or components thereof occurring on the material.Illustratively, the contaminants may be those known to cause toxicity,infectivity, pyrogenicity, irritation potential, reactivity, hemolyticactivity, carcinogenicity and/or immunogenicity.

[0017] Biocompatibility—refers to the ability of a material to pass thebiocompatibility tests set forth in International Standards Organization(ISO) Standard No. 10993 and/or the U.S. Pharmacopeia (USP) 23 and/orthe U.S. Food and Drug Administration (FDA) blue book memorandum No.G95-1, entitled “Use of International Standard ISO-10993, BiologicalEvaluation of Medical Devices Part-1: Evaluation and Testing.”Typically, these tests assay as to a material's toxicity, infectivity,pyrogenicity, irritation potential, reactivity, hemolytic activity,carcinogenicity, and/or immunogenicity. A biocompatible structure ormaterial when introduced into a majority of patients will not cause anadverse reaction or response. In addition, it is contemplated thatbiocompatibility can be effected by other contaminants such as prions,surfactants, oligonucleotides, and other biocompatibility effectingagents or contaminants.

[0018] Contaminant—refers to an unwanted substance on, attached to, orwithin a material. This includes, but is not limited to: bioburden,endotoxins, processing agents such as antimicrobial agents, blood, bloodcomponents, viruses, DNA, RNA, spores, fragments of unwanted tissuelayers, cellular debris, and mucosa.

[0019] Tela submucosa—refers to a layer of collagen-containingconnective tissue occurring under the mucosa in most parts of thealimentary, respiratory, urinary, integumentary, and genital tracts ofanimals.

[0020] The invention is generally directed to a medical device,comprising a collagenous biomaterial (also referred to as collagen-basedmatrices, tissue mucosa, tissue submucosa, intestines, biomaterial) andis further described in the non-limiting disclosure set forth below.

[0021] One such collagenous biomaterial includes tissue mucosa, whichalso further includes a tissue submucosa, which further includes a smallintestine submucosa (SIS), also described herein as tela submucosa. Telasubmucosa is a multi-laminate structure, comprising the tunicasubmucosa, lamina muscularis mucosa, and the stratum compactum.Collagenous materials can also have biotropic agents comprising at leastone of a proteoglycan, glycosaminoglycan, and growth factor. The SIS canbe made using the techniques described in Cook et al., WIPO PublicationWO 98/22158, dated May 28, 1998, which is the published application ofPCT/US97/14855, the disclosure of which is set forth below.

[0022] One type of collagenous mucosa is tela submucosa, and as withmany animal tissues, is generally aseptic in its natural state, providedthe human or animal does not have an infection or disease. This isparticularly the case since the tela submucosa is an internal layerwithin the alimentary, integumentary, respiratory, urinary, and genitaltracts of animals. Accordingly, it is generally not exposed to bacteriaand other cellular debris such as the epithelium of the intestinaltract. One feature of the present invention is the discovery that bydisinfecting the source tissue for the tela submucosa prior todelamination, the aseptic state of the tela submucosa layer can bepreserved or substantially preserved, particularly if the delaminationprocess occurs under sterile conditions.

[0023] Other sources of mucosa exist. For example, the mucosa can alsobe derived from vertebrate liver tissue as described in WIPOPublication, WO 98/25637, based on PCT application PCT/US97/22727; fromgastric mucosa as described in WIPO Publication, WO 98/26291, based onPCT application PCT/US97/22729; from stomach mucosa as described in WIPOPublication, WO 98/25636, based on PCT application PCT/US97/23010; orfrom urinary bladder mucosa as described in U.S. Pat. No. 5,554,389; thedisclosures of all are expressly incorporated herein.

[0024] In particular, it has been discovered that disinfecting the telasubmucosa source, followed by removal of a purified biomaterialincluding the tela submucosa, e.g. by delaminating the tela submucosafrom the tunica muscularis and the tunica mucosa, minimizes the exposureof the tela submucosa to bacteria and other contaminants. In turn, thisenables minimizing exposure of the isolated tela submucosa biomaterialto disinfectants or sterilants if desired, thus substantially preservingthe inherent biochemistry of the tela submucosa and many of the telasubmucosa's beneficial effects.

[0025] A tela submucosa implantable collagen biomaterial according tothe present invention can, as indicated above, be obtained from thealimentary, respiratory, urinary, integumentary, or genital tracts ofanimals. Preferably, the tela submucosa tissues, which arecollagen-based and thus predominantly collagen, are derived from thealimentary tract of mammals, such as cows, sheep, dogs, and mostpreferably from the intestinal tract of pigs. A most preferred source ofwhole small intestine is harvested from mature adult pigs weighinggreater than about 450 pounds. Intestines harvested from healthy,non-diseased animals will contain blood vessels and blood supply withinthe intestinal tract, as well as various microbes such as E. colicontained within the lumen of the intestines. Therefore, disinfectingthe whole intestine prior to delamination of the tela submucosasubstantially removes these contaminants and provides a preferredimplantable tela submucosa tissue which is substantially free of bloodand blood components as well as any other microbial organisms, pyrogensor other pathogens that may be present. In effect, this procedure isbelieved to substantially preserve the inherent aseptic state of thetela submucosa, although it should be understood that it is not intendedthat the present invention be limited by any theory. All that isrequired or be met is that the biomaterial satisfies the above-mentionedcriteria.

[0026] It is also desirable that the collagenous biomaterial accordingto the present invention be substantially free of any antiviral agentsor any antimicrobial type agents which can affect the biochemistry ofthe biomaterial and its efficacy upon implantation. In the past, onemethod of treating such tissue material is to rinse the delaminatedtissue in saline and soak it in an antimicrobial agent, for example, asdisclosed in U.S. Pat. No. 4,956,178. While such techniques canoptionally be practiced with isolated collagenous mucosa or submucosa ofthe present invention, preferred processes according to the presentinvention avoid the use of antimicrobial agents and the like which cannot only affect the biochemistry of the collagenous biomaterial but alsocan be unnecessarily introduced into the tissues of the patient.

[0027] As discussed above, it has been discovered that a highly pureform of an implantable tela submucosa collagen biomaterial can beobtained by first disinfecting a tela submucosa source prior to removinga purified collagen biomaterial including the tela submucosa layer, e.g.by delaminating the tela submucosa source. It has also been discoveredthat certain processing advantages as well as improved properties of theresultant tela submucosa layer are obtained by this process, includinggreater ease in removing attached tissues from the submucosa layer, anda characteristic, low contaminant profile.

[0028] Processes of the invention desirably involve first rinsing thetela submucosa source one or more times with a solvent, suitably water.The rinsing step is followed by treatment with a disinfecting agent. Thedisinfecting agent is desirably an oxidizing agent. Preferreddisinfecting agents are peroxy compounds, preferably organic peroxycompounds, and more preferably peracids. Such disinfecting agents aredesirably used in a liquid medium, preferably a solution, having a pH ofabout 1.5 to about 10, more preferably a pH of about 2 to about 6, andmost preferably a pH of about 2 to about 4. In methods of the presentinvention, the disinfecting agent will generally be used underconditions and for a period of time which provide the recovery ofcharacteristic, purified submucosa matrices as described herein,preferably exhibiting a bioburden of essentially zero and/or essentialfreedom from pyrogens. In this regard, desirable processes of theinvention involve immersing the tissue source (e.g. by submersing orshowering) in a liquid medium containing the disinfecting agent for aperiod of at least about 5 minutes, typically in the range of about 5minutes to about 40 hours, and more typically in the range of about 0.5hours to about 5 hours.

[0029] A preferred peroxy disinfecting agent is hydrogen peroxide. Theconcentration of hydrogen peroxide can range from about 0.05% to 30% byvolume. More preferably the hydrogen peroxide concentration is fromabout 1% to 10% by volume and most preferably from about 2% to 5% byvolume. The solution can or can not be buffered to a pH from about 5 to9. More preferably the pH is from about 6 to 7.5. These concentrationscan be diluted in water or in an aqueous solution of about 2% to about30% by volume alcohol. Most preferably the alcohol is ethanol. Thesolution temperature can range from about 15 to 50° C. More preferablythe solution temperature is from about 20 to 40° C. Most preferably, thesolution temperature is from about 32 to 37° C. The exposure time canrange from about 10 to 400 minutes. Preferably, the exposure time isfrom about 120 to 240 minutes. More preferably, the exposure time isfrom 180 to 210 minutes.

[0030] A preferred organic peroxide disinfecting agent is perpropionicacid. The concentration of perpropionic acid can range from about 0.1%to 10% by volume. More preferably the perpropionic acid concentration isfrom about 0.1% to 1.0% by volume and most preferably from about 0.2% to0.5% by volume. These concentrations of perpropionic acid can be dilutedin water or in an aqueous solution of about 2% to about 30% by volumealcohol. Most preferably the alcohol is ethanol. The tela submucosatissue source can be exposed to the organic peroxide solution forperiods from about 15 minutes to about 40 hours, and more typically inthe range of about 0.5 hours to about 8 hours. Other peroxy disinfectingagents are suitable for use as described in “Peroxygen Compounds”, S.Block, in Disinfection, Sterilization and Preservation, S. Block,Editor, 4th Edition, Philadelphia, Lea & Febiger, pp. 167-181, 1991; and“Disinfection with peroxygens”, M. G. C. Baldry and J. A. L. Fraser, inIndustrial Biocides, K. Payne, Editor, New York, John Wiley and Sons,pp. 91-116, 1988.

[0031] Another oxidizing disinfecting agent is chlorhexidine(1,6-di(4-chlorophenyldiguanido)hexane) in its digluconate form. Theconcentration of chlorhexidine digluconate can range from about 0.1% to15% by weight. More preferably, the chlorhexidine digluconateconcentration is from about 0.1% to 2% by weight and most preferablyfrom about 0.2% to 5% by weight. The solution can or can not be bufferedto a pH from about 5 to 8. More preferably the pH is from about 5.5 to7. These concentrations can be diluted in water or in an aqueoussolution of about 2% to about 20% by volume alcohol. Most preferably thealcohol is ethanol at a concentration of about 5% to 10%. The solutiontemperature can range from about 15 to 30° C. The exposure time canrange from about 10 to 400 minutes. More preferably the exposure time isfrom about 30 to 60 minutes. Other chlorine agents are described in“Chlorhexidine”, G. W. Denton, in Disinfection, Sterilization andPreservation, S. Block, Editor, 4th Edition, Philadelphia, Lea &Febiger, pp. 274-289, 1991.

[0032] In preferred preparative processes, a peracid or otherdisinfecting agent can be dissolved in a dilute aqueous alcoholsolution, preferably wherein the alcohol has from 1 to about 6 carbonatoms, and wherein the alcohol can generally comprise from about 1% toabout 30% by volume of the solution. More preferred alcohols for use inthe invention are selected from the group consisting of ethanol,propanols and butanols. Ethanol is a preferred alcohol for thesepurposes.

[0033] When a peracid is used in the disinfection, it is preferablyselected from the group consisting of peracetic acid, perpropionic acidor perbenzoic acid. Peracetic acid is the most preferred disinfectingagent. The peracetic acid is preferably diluted into about a 2% to about10% by volume alcohol solution. The concentration of the peracetic acidcan range, for example, from about 0.05% by volume to about 1.0% byvolume. Most preferably the concentration of the peracetic acid is fromabout 0.1% to about 0.3% by volume. Hydrogen peroxide can also be usedas a disinfecting agent. Alternatively, or in addition, the telasubmucosa tissue source, e.g. from small intestine, can be disinfectedutilizing disinfecting agents such as glutaraldehyde, formalin and thelike, which are also known for their ability to introduce substantialcrosslinking into collagen matrices, in contrast to the action of otherdisinfecting agents such as peracids which can be used to disinfectwithout introducing such crosslinking. Additionally, the tela submucosasource can be treated with radiation, e.g., gamma radiation, forpurposes of disinfection.

[0034] Variations on the disinfection process can also include thefollowing:

[0035] 1. Intestine is treated with 0.2% peracetic acid, 5% ethanolsolution at a ratio of 10:1 solution to intestine ratio by weight.Solution has a pH of 2.6. Solution and intestine are vigorously mixedfor two hours.

[0036] 2. Intestine is treated with 1% peracetic acid, 25% ethanolsolution at a ration of 5:1 solution to intestine ratio by weight.Solution has a pH of 2. Solution and intestine are vigorously mixed forone hour.

[0037] 3. Intestine is treated with 1% peracetic acid, 15% ethanol, and10% hydrogen peroxide solution at a ratio of 5:1 solution to intestineratio by weight. Solution and intestine are vigorously mixed for onehour.

[0038] 4. Whole small intestine is rinsed four times with high puritywater for 15 minutes. The intestine is then subjected to 1.5 MRADElectron Beam radiation.

[0039] 5. Whole small intestine is rinsed four times with high puritywater for 15 minutes. Lengthwise along a conveyor belt, the intestine issubjected to high-intensity pulsed light which disinfects the intestine.

[0040] Following the treatment as described above, the tela submucosalayer is delaminated from its source, e.g., whole intestine, cow uterusand the like. It has been found that by following thispost-disinfection-stripping procedure, it is easier to separate the telasubmucosa layer from the attached tissues, e.g. at least from attachedtunica muscularis tissue, as compared to stripping the tela submucosalayer prior to disinfection. Moreover it has been discovered that theresultant tela submucosa layer in its most preferred form exhibitssuperior histology, in that there is less attached tissue and debris onthe surface compared to a tela submucosa layer obtained by firstdelaminating the tela submucosa layer from its source and thendisinfecting the layer. Moreover, a more uniform tela submucosa tissuecan be obtained from this process, and a tela submucosa having the sameor similar physical and biochemical properties can be obtained moreconsistently from each separate processing run. Importantly, a highlypurified, substantially sterile tela submucosa is obtained by thisprocess. The stripping of the tela submucosa source is preferablycarried out by utilizing a disinfected or sterile casing machine, toproduce a tela submucosa which is substantially sterile and which hasbeen minimally processed. A suitable casing machine is the Model 3-U-400Stridhs Universal Machine for Hog Casing, commercially available fromthe AB Stridhs Maskiner, Götoborg, Sweden. Therefore, the measuredbioburden levels are minimal or substantially zero. Of course, othermeans for delaminating the tela submucosa source can be employed withoutdeparting from the present invention, including for example those meanswell known in the art, including delaminating by hand.

[0041] It has also been discovered that more preferred processesaccording to the present invention, not only will eliminate orsignificantly reduce contaminants contained in the tela submucosacollagen biomaterial, but also will produce a tissue which exhibits nosubstantial degradation of physical and mechanical properties, e.g.,differential porosity (i.e. wherein one side of the submucosa layer hasgreater porosity than the other side), and good strength, for exampleburst strength. Also, it has been discovered that more preferredprocesses do not affect the differential porosity of the tela submucosacollagen biomaterial, which ultimately affects the level of efficacy ofthis tissue implant. For example, the tissue is not necessarily treatedwith a crosslinking agent or a material that disrupts the porosity orinherent, native structure of the collagen biomaterial. Moreover, whenhydrogen peroxide is employed, the biomaterial as a whole has greaterporosity as well as a higher oxygen content. This helps to ensure theabsence of contaminants e.g., endotoxins, pyrogens, and the like.

[0042] Preferred collagen-based matrices of the invention, preferablysubmucosa-containing matrices, are also characterized by the lowcontaminant levels set forth in Table 1 below, each contaminant leveltaken individually or in any combination with some or all of the otherdisclosed contaminant levels. The abbreviations in Table 1 are asfollows: CFU/g=colony forming units per gram; PFU/g=plaque forming unitsper gram; μg/mg=micrograms per milligram; ppm/kg=parts per million perkilogram; and EU/g=endotoxin units per gram. TABLE 1 FIRST SECOND THIRDPREFERRED PREFERRED PREFERRED FEATURE LEVEL LEVEL LEVEL ENDOTOXIN <12EU/g <10 EU/g <5 EU/g BIOBURDEN <2 CFU/g <1 CFU/g <0.5 CFU/g FUNGUS <2CFU/g <1 CFU/g <0.5 CFU/g NUCLEIC ACID <10 μg/mg <5 μg/mg <2 μg/mg VIRUS<500 PFU/g <50 PFU/g <5 PFU/g PROCESSING <100,000 <1,000 <100 AGENTppm/kg ppm/kg ppm/kg

[0043] Even more preferred collagen-based matrices of the inventioncontain an endotoxin level of less than 1 EU/g, and most preferably lessthan 0.5 EU/g.

[0044] Purified collagen-based matrices according to the presentinvention can be processed in a number of ways, to provide collagenousmatrices useful both in vitro and in vivo. For example, the submucosacan be configured to provide tissue grafts useful in vascularapplications, e.g., as generally described in U.S. Pat. No. 2,127,903and 4,902,508.

[0045] The tela submucosa of the invention possesses mechanicalproperties highly desirable for tissue graft materials in vascularapplications, including low porosity index, high compliance, and a highburst strength. One skilled in the art will appreciate that thepreferred tissue graft material will be of low enough porosity toprevent intraoperative hemorrhage and yet of high enough porosity toallow extension of a newly-developed vasa vasorum through the graftmaterial to nourish the neointimal and luminal surface.

[0046] Tela submucosa tissue of the present invention can also beprocessed to provide fluidized compositions, for instance usingtechniques as described in U.S. Pat. No. 5,275,826. In this regard,solutions or suspensions of the tela submucosa can be prepared bycomminuting and/or digesting the tela submucosa with a protease (e.g.trypsin or pepsin), for a period of time sufficient to solubilize thetissue and form substantially homogeneous solution. The submucosastarting material is desirably comminuted by tearing, cutting, grinding,shearing or the like. Grinding the submucosa in a frozen or freeze-driedstate is advantageous, although good results can be obtained as well bysubjecting a suspension of pieces of the submucosa to treatment in ahigh speed blender and dewatering, if necessary, by centrifuging anddecanting excess waste. The comminuted tela submucosa can be dried, forexample freeze dried, to form a powder. Thereafter, if desired, thepowder can be hydrated, that is, combined with water or buffered salineand optionally other pharmaceutically acceptable excipients, to form afluid tissue graft composition, e.g. having a viscosity of about 2 toabout 300,000 cps at 25EC. The higher viscosity graft compositions canhave a gel or paste consistency.

[0047] Fluidized tela submucosa of this invention finds use as aninjectable heterograft for tissues, for example, bone or soft tissues,in need of repair or augmentation most typically to correct trauma ordisease-induced tissue defects. The present fluidized submucosacompositions are also used advantageously as a filler for implantconstructs comprising, for example, one or more sheets of tela submucosaformed into sealed (sutured) pouches for use in cosmetic ortrauma-treating surgical procedures.

[0048] In one illustrative preparation, tela submucosa prepared asdescribed herein is reduced to small pieces (e.g. by cutting) which arecharged to a flat bottom stainless steel container. Liquid nitrogen isintroduced into the container to freeze the specimens, which are thencomminuted while in the frozen state to form a coarse tela submucosapowder. Such processing can be carried out, for example, with a manualarbor press with a cylindrical brass ingot placed on top of the frozenspecimens. The ingot serves as an interface between the specimens andthe arbor of the press. Liquid nitrogen can be added periodically to thetela submucosa specimens to keep them frozen.

[0049] Other methods for comminuting tela submucosa specimens can beutilized to produce a tela submucosa powder usable in accordance withthe present invention. For example, tela submucosa specimens can befreeze-dried and then ground using a manual arbor press or othergrinding means. Alternatively, tela submucosa can be processed in a highshear blender to produce, upon dewatering and drying, a tela submucosapowder.

[0050] Further grinding of the tela submucosa powder using a prechilledmortar and pestle can be used to produce a consistent, more finelydivided product. Again, liquid nitrogen is used as needed to maintainsolid frozen particles during final grinding. The powder can be easilyhydrated using, for example, buffered saline to produce a fluidizedtissue graft material of this invention at the desired viscosity.

[0051] To prepare another preferred fluidized material, a tela submucosapowder can be sifted through a wire mesh, collected, and subjected toproteolytic digestion to form a substantially homogeneous solution. Forexample, the powder can be digested with 1 mg/ml of pepsin (SigmaChemical Co., St. Louis, Mo.) and 0.1 M acetic acid, adjusted to pH 2.5with HCl, over a 48 hour period at room temperature. After thistreatment, the reaction medium can be neutralized with sodium hydroxideto inactivate the peptic activity. The solubilized submucosa can then beconcentrated by salt precipitation of the solution and separated forfurther purification and/or freeze drying to form a protease-solubilizedintestinal submucosa in powder shape.

[0052] Fluidized tela submucosa compositions of this invention find wideapplication in tissue replacement, augmentation, and/or repair. Thefluidized submucosal compositions can be used to induce regrowth ofnatural connective tissue or bone in an area of an existent defect. Byinjecting an effective amount of a fluidized submucosa composition intothe locale of a tissue defect or a wound in need of healing, one canreadily take advantage of the biotropic properties of the telasubmucosa. Interestingly, fluidizing SIS by comminution or enzymaticdegradation does not result in any appreciable loss of biotropicactivities, as shown in U.S. Pat. No. 5,275,826.

[0053] It is also possible to shape large surface area constructs bycombining two or more tela submucosa segments of the invention, forinstance using techniques as described in U.S. Pat. No. 2,127,903 and/orInternational Publication No. WO 96/32146, dated Oct. 17, 1996,publishing International Application No. PCT/US96/04271, filed Apr. 5,1996. Thus, a plurality of tela submucosa strips can be fused to oneanother, for example by compressing overlapping areas of the stripsunder dehydrating conditions, to form an overall planar construct havinga surface area greater than that of any one planar surface of theindividual strips used to shape the construct. Shapes can be made byusing sutures, staples, biocompatible adhesives such as collagen bindingpastes, or dehydrating overlapping structures then heating the structureas described in U.S. Pat. No. 3,562,820.

[0054] As described herein, the invention can take many shapes, such ascoiled, helical, spring-like, randomized, branched, sheet-like, tubular,spherical, fragmented, fluidized, comminuted, liquefied, suspended,gel-like, injectable, powdered, ground, sheared, and solid materialshape.

[0055] The tela submucosa powder can be used alone, or in combinationwith one or more additional bioactive agents such as physiologicallycompatible minerals, growth factors, antibiotics, chemotherapeuticagents, antigen, antibodies, enzymes, and hormones. Preferably, thepowder-form implant will be compressed into a predetermined,three-dimensional shape, which will be implanted into the bone regionand will substantially retain its shape during replacement of the graftwith endogenous tissues.

[0056] Tela submucosa of the invention can also be used as a cell growthsubstrate, illustratively in sheet, paste or gel shape in combinationwith nutrients which support the growth of the subject cells, e.g.eukaryotic cells such as endothelial, fibroblastic, fetal skin,osteosarcoma, and adenocarcinoma cells (see, e.g. InternationalPublication No. WO 96/24661 dated Aug. 15, 1996, publishingInternational Application No. PCT/US96/01842 filed Feb. 9, 1996). Incertain forms, the tela submucosa substrate composition will support theproliferation and/or differentiation of mammalian cells, including humancells.

[0057] The inventive tela submucosa can also serve as a collagenousbiomaterial in compositions for producing transformed cells, (see, e.g.,International Publication No. WO 96/25179, dated Aug. 22, 1996,publishing International Application No. PCT/US96/02136 filed Feb. 16,1996; and International Publication No. WO 95/22611 dated Aug. 24, 1995,publishing International Application No. PCT/US95/02251 filed Feb. 21,1995). Such compositions for cell transformation will generally includepurified tela submucosa of the present invention, for example influidized or paste shape as described in U.S. Pat. No. 5,275,826, incombination with a recombinant vector (e.g. a plasmid) containing anucleic acid sequence with which in vitro or in vivo target cells are tobe genetically transformed. The cells targeted for transformation caninclude, for example, bone progenitor cells.

[0058] In order to promote a further understanding of the presentinvention and its features and advantages, the following specificExamples are provided. It will be understood that these specificExamples are illustrative, and not limiting, of the present invention.

EXAMPLE 1

[0059] Thirty feet of whole intestine from a mature adult hog is rinsedwith water. This material is then treated in a 0.2% by volume peraceticacid in a 5% by volume aqueous ethanol solution for a period of twohours with agitation. The tela submucosa layer is then delaminated in adisinfected casing machine from the whole intestine. The delaminatedtela submucosa is rinsed four (4) times with sterile water and testedfor impurities or contaminants such as endotoxins, microbial organisms,and pyrogens. The resultant tissue was found to have essentially zerobioburden level. The tela submucosa layer separated easily andconsistently from the whole intestine and was found to have minimaltissue debris on its surface.

EXAMPLE 2

[0060] A ten foot section of porcine whole intestine is washed withwater. After rinsing, this section of tela submucosa intestinal collagensource material is treated for about two and a half hours in 0.2%peracetic acid by volume in a 5% by volume aqueous ethanol solution withagitation. Following the treatment with peracetic acid, the telasubmucosa layer is delaminated from the whole intestine. The resultanttela submucosa is then rinsed four (4) times with sterile water. Thebioburden was found to be essentially zero.

EXAMPLE 3

[0061] A small section of the tela submucosa intestinal collagenmaterial was subcutaneously implanted in a rat. Within 72 hours,significant angiogenesis was observed.

EXAMPLE 4

[0062] Two sections of small intestine are processed by differingmethods. The first section is rinsed in tap water, disinfected for 2hours in a 5% by volume aqueous ethanol solution comprising 0.2% byvolume peracetic acid, pH approximately 2.6, delaminated to the telasubmucosa, rinsed in purified water, divided into two samples andrapidly frozen. The second section is rinsed in tap water, delaminatedto the tela submucosa, rinsed in purified water, placed in a 10%neomycin sulfate solution for 20 minutes (as described in U.S. Pat. No.4,902,508), rinsed in purified water, divided into two samples andrapidly frozen. The four above-prepared samples are tested for bioburdenand endotoxin levels. The first two samples each have bioburdens of lessthan 0.1 CFU/g and endotoxin levels of less than 0.1 EU/g. The secondtwo samples have respective bioburdens of 1.7 CFU/g and 2.7 CFU/g andrespective endotoxin levels of 23.9 EU/g and 15.7 EU/g.

EXAMPLE 5

[0063] Three sections of small intestine are processed by differingmethods. The first is rinsed in tap water, disinfected for 2 hours in a5% by volume aqueous ethanol solution comprising 0.2% by volumeperacetic acid, pH about 2.6, delaminated to the tela submucosa, rinsedin purified water, and rapidly frozen. The second is rinsed in tapwater, delaminated to the tela submucosa, rinsed in purified water,disinfected according to the methods of Example 1 in U.S. Pat. No.5,460,962 (treatment for 40 hours in a 0.1% by volume aqueous solutionof peracetic acid, buffered to pH 7.2), and rapidly frozen. The third isrinsed in tap water, delaminated to the tela submucosa, rinsed inpurified water, disinfected according to the methods of Example 2 inU.S. Pat. No. 5,460,962 (treatment in 0.1% by volume peracetic acid inhigh salt solution, buffered to pH 7.2), and rapidly frozen. All threesamples were tested for endotoxins. The endotoxin levels were <0.14 EU/gfor the first sample, >24 EU/g for the second sample, and >28 EU/g forthe third sample.

EXAMPLE 6

[0064] Two sections of porcine small intestine were infected with 7×10⁶plaque forming units (PFU) of virus. Both were exposed to a 0.18%peracetic acid, 4.8% aqueous ethanol solution at a nine-to-one weightratio of solution to material. A first sample was immersed in thissolution for 5 minutes; the second was immersed for 2 hours. Thematerial processed for 5 minutes exhibited 400 PFU per gram of material.The material processed for 2 hours exhibited zero PFU per gram ofmaterial.

EXAMPLE 7

[0065] Purified tela submucosa, prepared as described herein, was testedto determine its nucleic acid content. Four samples of material weighing5 mg each were subjected to DNA/RNA extraction as detailed in theDNA/RNA Isolation Kit by Amersham Lifescience Inc., Arlington Heights,Ill. Nucleic acid quantitation was performed by spectrophotometricdetermination of solution optical densities at 260 nm and 280 nm. Theaverage nucleic acid content was 1.9±0.2 mg per milligram of material.

[0066] Small intestinal submucosa, prepared as described by U.S. Pat.No. 4,902,508, was tested to determine its nucleic acid content. Foursamples of material weighing 5 mg each were subjected to DNA/RNAextraction as detailed in the DNA/RNA Isolation Kit by Amersham. Nucleicacid quantitation was performed by spectrophotometric determination ofsolution optical densities at 260 nm and 280 nm. The average nucleicacid content was 2.4±0.2 mg per milligram of material.

EXAMPLE 8

[0067] Sections of tela submucosa prepared according to the methodsdescribed herein were sent to an independent testing laboratory (NAmSA,Inc., Northwood, Ohio) for biocompatibility testing as described in thestandard ISO 10993. The samples were tested for USP Acute SystemicToxicity, USP Intracutaneous Toxicity, Cytotoxicity, LAL Endotoxin,material-mediated Pyrogenicity, Direct Contact Hemolysis, and PrimarySkin Irritation. The samples passed all tests, indicating that thematerial is biocompatible.

EXAMPLE 9

[0068] Using the procedure set forth in U.S. Pat. No. 5,460,962, twosamples were analyzed. The first Kemp sample indicated an endotoxinlevel greater than 24 endotoxin units per gram and the second Kempsample indicated an endotoxin level greater than 28 endotoxin units pergram. Thus, when using the procedure set forth in Kemp '962, theendotoxin levels fall outside the biocompatibility levels.

EXAMPLE 10

[0069] Using the procedures set forth in U.S. Pat. Nos. 4,902,508 and5,372,821 issued to Badylak, the endotoxin level shown ranges as high as23.9 endotoxin units per gram per sample. This falls outside thepermissible range and thus does not the meet the criteria ofbiocompatibility. The invention, prepared in the above prescribed mannerof disinfection first then delamination, was observed to have anendotoxin level of less than 12 endotoxin units per gram, and moreparticularly, reported an endotoxin level of less than 5 endotoxin unitsper gram. Thus, the material of the present invention is biocompatibleas defined above.

EXAMPLE 11

[0070] With reference to FIGS. 2A and 2B, in preparing a lyophilized SISsponge 10, comminuted SIS (with a mean particle size of approximately150 μm) was centrifuged in a Beckman TJ-6 centrifuge with a speed of1550×g for 15 minutes. The supernatant was poured off leaving adough-like consistency of SIS remaining. The material was then pouredinto various polycarbonate molds and frozen at cold temperatures,preferably at −80° C. for at least 2 hours. The material was thenvacuum-dried for 6 hours in a lyophilizing system with the condenser at−70° C. at a vacuum pressure of less than 100 millitorr, preferably 15millitorr. Optionally, the sponge 10 can then be sterilized usingethylene oxide. The resulting structure was an SIS sponge 10, having acreamyish yellow-white color.

[0071] In varying the above procedures, the following attributes werenoticed. Varying the “g” force from 0-15000×g, centrifugation at thehigher “g” results in an increased sponge 10 density, a decreased spongeabsorbency, and increased tensile strength. If the material weresterilized by radiation instead, for example using 10-25 kGy, noted wasa decrease in tensile strength. Using comminuted hydrated SIS with apossible fragment size of 25 to 3000 μm produced a more condensed,higher density sponge, whereas larger fragments produced a less denselarger pore sized sponge. Adding sugar molecules to the comminutedhydrated SIS resulted in a increased pore size sponge. Furthermore, theaddition of cross-linking agents, such as DANECOL, carbodiimide, orglutaraldehyde, caused an increased tensile strength that did not breakdown as easily upon the addition of water. In addition, moderating thepH of the suspension, from 2-10, preferably 3-5, demonstrated that ahigher pH will reduce the tensile strength of the sponge. Since theresulting sponge can take the shape of the mold in which it is placed,modifying the mold shape will modify the sponge shape.

[0072] The resulting sponge finds applicability, inter alia, inproviding hemostasis in a large wound; fills a large defect; provides athree dimensional structure for cell culture or in vivo growth; providesa plug to stop bleeding; can be an injectable form; can be used forvessel embolization. EXAMPLE 12 In accordance with the presentinvention, the lyophilized SIS was compared to other forms of SIS toprovide the following data: Non Single De- De- 4-Layered layered wateredwatered 4-Layered Vacuum- Lyophilized SIS SIS Lyophilized Pressed Sheetsponge Sponge Sheet Sheet** Absorbency 5.3 5.6 4.2 3.1 1.0 Level (gwater per g of sample)*** Density .26 .16 .20 .26 .65 (g/cm³) Load at882 161 587 4501 5931 Failure of 1 cm wide strip (gf) Stress at 7737 571792 11,169 237,240 failure (gf/cm²)

[0073] Notably, with reference to FIG. 1, the increasing number oflyophilized sheets 15 used indicated a decrease in the absorbency levelto approximately 1.01 grams of water per gram of sample. Furthermore, byincreasing the amount of water centrifuged out, the density increased.

EXAMPLE 13

[0074] With reference to FIG. 1, to prepare lyophilized multilaminatesheets 15, a sheet 20 of hydrated SIS was laid down on a non-sticksurface, such as polytetrafluoroethylene (TEFLON(R)) and smoothed toremove any ripples or entrapments between the sheet 20 and the non-sticksurface. A second sheet was placed over the first sheet and smoothed outin the same fashion. The process was repeated until the preselectednumber of sheets 20 were stacked forming the multilaminate sheetstructure 15. A thin layer of high purity water was glazed over the topsheet of SIS and the material was frozen at −80° C. for at least 2hours. The material was then vacuum dried at a vacuum pressure less than100 millitorr (preferably less than 15 millitorr) in a condenseroperating at −70° C. for six hours. The dried sheets were then cut intoa preselected shape, sealed in packaging, and terminally sterilizedusing ethylene oxide.

[0075] By modifying the process in the manner described in Example 11,the sheet may have different attributes. For example, it was noted thatadjusting the pH of the hydrated SIS to between 2-10, preferably 3-5,demonstrated an increased lamination strength. In addition, a tissueglue may be present between the sheets to increase lamination strength.

EXAMPLE 14

[0076] In making the SIS into various forms and lyophilizing theproduct, it was noted that the porosity index of the SIS changed. Asheet of hydrated SIS was attached to the bottom of a water columnexerting a hydrostatic pressure of approximately 35 inches. The amountof water collected via pass through after 30 seconds was measured. Theexperiment was repeated using a single sheet of lyophilized SIS underthe water column. In comparing the hydrated form of a single sheet ofSIS with the lyophilized form, the hydrated form had a mean of 9.59grams of water collected, with a standard deviation of 5.22. Thelyophilized form had a mean of 0.2 grams of water with a standarddeviation of 0.13.

[0077] With reference to FIGS. 2A and 2B, as with the lyophilizedproduct, the product can be packaged in packages that are, inter alia,gas permeable, hermetically sealed, sterile, sealed, UV protected,double pouched, or some combination thereof. By UV barriered or UVprotected, it is meant that the packages or pouches are made of amaterial that substantially blocks the contents from UV radiation. Forexample, the package can comprise the material disclosed in U.S. Pat.No. 5,489,022, which issued on Feb. 06, 1996 to Sabin Corp.(Bloomington, Ind.), the disclosure of which is entirely incorporated byreference herein. Preferably, the product is packaged in gas permeabledouble pouched packages that have a UV barrier to protect the internalproduct by placing the product in the package and sealing it. Gaspermeable packages are desirable as it facilitates terminalsterilization with ethylene oxide. A double pouched package system 25 isdesirable in that it permits the user to open the exterior pouch 30 anddrop the inner pouch 35 on sterile surfaces. However, the packaging mayinclude a plurality of pouches nested within each other, such that thereis a pouch within a pouch within a pouch, etc. with the biomaterialbeing in the inner or innermost pouch. By sterile, it is meant that thepackage is sterilized and is of such construction and material as tokeep the contents of the package and inner pouches sterile. The packagecan also contain a buffered solution 50 to keep the product wet. Thesolution can also contain the pharmocologic agents described above topermit long-term infiltration of the agent into the collagenousbiomaterial product. The solution chosen can also include thecharacteristic that the final package can be terminally sterilized andthus sterilize the solution without deleteriously changing the solutioncharacteristics.

[0078] In another embodiment of the invention, the medical deviceproduct comprises a submucosa of a warm blooded vertebrate made in themanner described above. The submucosa has an endotoxin level less than12 endotoxin units per gram of submucosa and the submucosa furtherhaving an absorbency level of greater than 1.01 grams of water per gramof submucosa. The collagenous biomaterial further made by the processcomprising the steps of sterilizing a source of submucosal tissue fromthe sources identified above; delaminating the source of submucosaltissue to expose the submucosa; freezing the submucosa in the mannersdescribed above; and drying the submucosa under vacuum pressure.Optionally, the collagenous biomaterial may be comminuted by grinding,shearing, or fragmenting the biomaterial in the manner described above.In addition, the collagenous biomaterial can further include packagingthe biomaterial in at least one of a gas permeable, sealed, hermeticallysealed, sterile, UV protected, and a plurality of pouches.

[0079] The collagen biomaterial can be made radiopaque by a variety ofconventional procedures, none of which has yet been applied to telasubmucosa. In one embodiment of the invention, the collagen material hasa shape, namely made into sheets, either in lyophilized ornon-lyophilized form. With reference to FIGS. 1, 2A, and 2B, anyradiopaque substance 40, including but not limited to, tantalum such astantalum powder, can be spread along the surface of the tela submucosa,such as on the serosal side. Other radiopaque materials 40 comprisebismuth and barium, including but not limited to, bismuth oxychlorideand barium sulphate, as well as other conventional markers. As usedherein, the term “disposed” on shall be construed to include disposedon, disposed throughout, disposed in, disposed with, disposed alongwith, applied on, applied with, applied through, applied in, applied inconjunction with, and the like. With particular reference to telasubmucosa, the differential porosity of the material can enable moreradiopaque material 40 to be disposed on the tela submucosa.

[0080] In one particular embodiment, radiopaque marker tantalum powderwas disposed on a sheet of tela submucosa by rubbing it onto the serosalside of the tela submucosa. The tela submucosa was then made intovarious shapes, such as, but not limited to, having the shape of abrush-like, braided, branched, helical, spherical, cubic, cylindrical,tubular, injectable, randomized, layered, and sheet-like shapes. Forexample, an injectable shape of the invention can be readily made bycomminuting the invention into small fibrils, fragments, or the like,then suspending them in solution, such as, but not limited to, abiocompatible gelatin suspension. Due to the viscosity of the gelatinsuspension, the invention, when injected into the lumen of an aneurysm,will stay in the lumen and provide the therapeutic benefit to theaneurysm.

[0081] The invention and collagenous biomaterial can be made in layers.In this manner, the collagenous material can increase its structuralintegrity, strength, and applicability. In one embodiment of theinvention, a dual layer of collagenous biomaterial can be used in sheetsand either the radiomarker or pharmacologic agent, or both can bedisposed in between the layers.

[0082] In similar fashion, the pharmacologic agent 45 can be disposed onthe collagenous material. As used herein, the pharmacologic agent 45includes, but is not limited to, growth factors, proteins,proteoglycans, glycosaminoglycans, physiological compatible minerals,antibiotics, chemotherapeutic agents, enzymes, drugs, and hormones. Theagent can be disposed on the serosal or mucosal sides of the submucosa,or can be disposed on the same or opposite sides of the collagenousmaterial. Consideration of placement can be important depending on thedesired shape of the final device. For example, where the shape istubular, it can be desirable to place the pharmacologic agent 45 on theabluminal surface since that surface will be in contact with thesurrounding tissue. On the other hand, if systemic release of the agent45 is contemplated, then the agent can be placed on the lumenal side topermit the blood to contact the agent and carry it away. Utilizing thebraid shape, each individual strip can be treated with different agents.

[0083] It will be appreciated that variations of the above-describedprocessing procedures are intended to be within the scope of thisinvention. For example, the source tissue for the collagenousbiomaterial, e.g., stomach, whole intestine, cow uterus and the like,can be partially delaminated, treated with a disinfecting or sterilizingagent followed by complete delamination of the tela submucosa.Illustratively, attached mesentery layers, and/or serosa layers of wholeintestine can be advantageously removed prior to treatment with thedisinfecting agent, followed by delamination of remaining attachedtissues from the tela submucosa These steps can or can not be followedby additional disinfection steps, e.g., enzymatic purification and/ornucleic acid removal. Alternatively, the tela submucosa source can beminimally treated with a disinfecting or other such agent, the telasubmucosa delaminated from the tunica muscularis and tunica mucosa,followed by a complete disinfection treatment to attain the desiredcontaminant level(s). All such variations and modifications arecontemplated to be a part of the process described herein and to bewithin the scope of the invention.

[0084] Many alterations and modifications can be made by those ofordinary skill in the art without departing from the spirit and scope ofthe invention. The illustrated embodiments have been shown only forpurposes of clarity and examples, and should not be taken as limitingthe invention as defined by the appended claims, which include allequivalents, whether now, or later devised.

What is claimed is:
 1. A collagenous biomaterial medical device,comprising: a) a submucosa tissue; b) the submucosa having an endotoxinlevel less than 12 endotoxin units per gram per gram of submucosa; andc) the submucosa having an absorbency level of greater than 1.01 gramsof water per gram of submucosa.
 2. The collagenous biomaterial of claim1 wherein the submucosa has an absorbency level of greater than 2.0grams of water per gram of submucosa.
 3. The collagenous biomaterial ofclaim 1 wherein the submucosa has an absorbency level of greater than3.0 grams of water per gram of submucosa.
 4. The collagenous biomaterialof claim 1 wherein the submucosa has an absorbency level of greater than3.1 grams of water per gram of submucosa.
 5. The collagenous biomaterialof claim 1 wherein the submucosa has an absorbency level of greater than5.3 grams of water per gram of submucosa.
 6. The collagenous biomaterialof claim 1 wherein the submucosa has an absorbency level approximatelyin the range of 3.0 to 5.4 grams of water per gram of submucosa.
 7. Thecollagenous biomaterial of claim 6 wherein a radiopaque marker isdisposed on the submucosa.
 8. The collagenous biomaterial of claim 1wherein the submucosa has a density of greater than 0.01 grams per cubiccentimeter.
 9. The collagenous biomaterial of claim 1 wherein thesubmucosa has a density less than 0.65 grams per cubic centimeter. 10.The collagenous biomaterial of claim 1 wherein the submucosa has adensity greater than 0.10 grams per cubic centimeter and less than 0.65grams per cubic centimeter.
 11. The collagenous biomaterial of claim 1wherein the submucosa has a density greater than 0.15 grams per cubiccentimeter and less than 0.65 grams per cubic centimeter.
 12. Thecollagenous biomaterial of claim 1 wherein the submucosa has a densitygreater than 0.16 and less than 0.65 grams per cubic centimeter.
 13. Thecollagenous biomaterial of claim 1 wherein the submucosa has a densitygreater than 0.20 and less than 0.65 grams per cubic centimeter.
 14. Thecollagenous biomaterial of claim 1 wherein the submucosa has a densitybetween 0.10 and 0.40 grams per cubic centimeter.
 15. The collagenousbiomaterial of claim 1 wherein the submucosa has a density between 0.10and 0.30 grams per cubic centimeter.
 16. The collagenous biomaterial ofclaim 15 wherein a radiopaque marker is disposed on the submucosa. 17.The collagenous biomaterial of claim 1 further comprising a packagecontaining the biomaterial.
 18. The collagenous biomaterial of claim 17wherein the package further comprises at least one of a gas permeable,sealed, hermetically sealed, sterile, UV protected, and a plurality ofpouches.
 19. The collagenous biomaterial of claim 17 wherein the packagecomprises a double pouched, gas permeable, and UV protected package. 20.A collagenous biomaterial medical device, comprising a submucosa of awarm blooded vertebrate, the submucosa having an endotoxin level lessthan 12 endotoxin units per gram of submucosa, the submucosa furtherhaving an absorbency level of greater than 1.01 grams of water per gramof submucosa, the collagenous biomaterial further made by the processcomprising the steps of: sterilizing a source of submucosal tissue;delaminating the source of submucosal tissue to expose the submucosa;freezing the submucosa; and drying the submucosa under vacuum pressure.21. The collagenous biomaterial of claim 20, wherein the step ofdelaminating includes the step of comminuting the exposed submucosa. 22.The collagenous biomaterial of claim 20 further including the step ofpackaging the biomaterial in at least one of a gas permeable, sealed,hermetically sealed, sterile, UV protected, and a plurality of pouches.23. The collagenous biomaterial of claim 22 further including the stepof packaging the biomaterial in a package comprising a gas permeable,sterile, UV protected package.
 24. The collagenous biomaterial of claim23 further including the step of packaging the biomaterial in a packagecomprising a plurality of pouches.
 25. A collagenous biomaterial medicaldevice, comprising: a) a submucosa tissue b) the submucosa having anendotoxin level less than 12 endotoxin units per gram of submucosa; c)the submucosa having an absorbency level of greater than 1.01 grams ofwater per gram of submucosa; d) the submucosa having a density greaterthan 0.10 grams per cubic centimeter and less than 0.65 grams per cubiccentimeter; and e) a package containing the submucosa.
 26. Thecollagenous biomaterial of claim 25, wherein a pharmacologic agent and aradiopaque marker are disposed on the submucosa.